Manual and Fully Automated Chemotaxis-Based Cancer Screening Yield Equivalent Performance: A Nine-Month Real-World,...

Background/Objectives: The N-NOSE test is a non-invasive, urine-based multi-cancer screening assay that uses Caenorhabditis elegans chemotaxis toward cancer-associated volatile organic compounds in human urine. Scaling the test from a manual research-grade workflow to a high-throughput clinical service has required automation, and the central question this raises, centering around whether mechanization alters the analytical performance of the test, must be answered with operational, not bench-top, data. Methods: Here, we present a nine-month (January-September 2023) real-world, side-by-side comparison of the two workflows operating under their actual routine clinical laboratory conditions: the manual chemotaxis assay performed by trained technicians at the Fukuoka Research and Development Center (R&D) and the fully automated Chemotaxis Scoring Apparatus (CSA) running continuously at the Tokyo Testing Center. Results: The manual workflow generated 551 paired chemotaxis index (CI) measurements from positive-control (PC) and negative-control (NC) synthetic urine/volatile organic compound (VOC)-mimic reference materials at each of two standard urine dilutions (10^-1 and 10^-2); over the same period, the CSA processed 2448 quality control samples (612 per control type) with both biobank-derived urine-based comparison materials and synthetic volatile organic compound reference standards. Both workflows produced large, highly significant, and quantitatively comparable PC-versus-NC separation under genuine operating conditions (manual: Δ_CI = 0.096 and 0.103; Welch's t = 19.83 and 21.95; p < 0.0001; Cohen's d = 1.19 and 1.32; CSA risk scale Δ_P-N = 14.47 with biobank-derived urine-based materials and 10.17 with synthetic VOC standards). The CSA risk score is a linear, monotonic transformation of the CI. Standardized separation is directly comparable across workflows and is concordant (Cohen's d: manual 1.19-1.32; CSA 0.80-1.44, all large); the manual and automated processes therefore show no meaningful difference in discriminative performance. Because the CSA mechanizes only the handling of worms, samples, and machine-vision counting around an unchanged biological transducer, the live nematode, analytical equivalence is the predicted outcome, and these data confirm it at scale in a real clinical laboratory setting. Conclusions: Automation of the N-NOSE process does not compromise its ability to discriminate cancer from non-cancer urine. These results provide real-world evidence supporting the validity, reproducibility, and reliability of the N-NOSE testing process and large-scale validation studies.